Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

Staphylococcus aureus is commonly found as normal flora on human skin. As such, it is a frequent cause of skin and soft tissue infection that often spreads to deeper tissue. It has become an especially important microorganism in both clinical and community settings due to its propensity to acquire resistance to antibiotics as well as the invasive mechanisms it utilizes. Specifically, community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) strains have developed resistance to beta-lactam antibiotics and, perhaps more importantly, many invasive pathogenic mechanisms. Thisdissertation explores novel virulence mechanisms of CA-MRSA infections through characterization of novel protein within the prevalent CA-MRSA strain, USA300. The main objective of thisdissertation is characterization of SAUSA300_1759 from the CA-MRSA strain USA300, which will establish this hypothetical protein as a critical virulence protein in most CA-MRSA strains of S. aureus. USA300 is responsible for most of the CA-MRSA cases in the United States. The purpose of this research was to identify the role in pathogenesis of this protein and determine its role in the infection cycle with the potential to identify new targets for possible treatment and therapy of MRSA infections. Bioinformatics analysis generated results indicating that homologous hypothetical proteins in many other MRSA strains, most of which were CA-MRSA. There were no results for homologous characterized proteins indicating the specific role this gene plays is a unique virulence mechanism in MRSA. SAUSA300_1759 has been successfully cloned, expressed, and purified for use in testing toxicity to Caenorhabditis elegans, a nematode used as a model organism to test bacterial toxin function. The toxicity tests resulted in death of the nematodes at an average time of seven minutes for the wild-type (WT) C. elegans . Mutant C. elegans strains, with gene mutations causing delayed apoptosis, had an average death time of 15–18 minutes. Killing assays comparing USA300 WT to a strain with a gene knockout of SAUSA300_1759 resulted in WT and mutant C. elegans deaths within consistent time frames established in previous work: there did not seem to be a significant difference between the WT and gene knockout USA300 strains effect on the death rates. C. elegans mutant strain, IM222, has a genetic mutation causing a variation in clumping, bordering, and hyperactivity phenotype compared to that of the WT strain. These behaviors of clumping and bordering are exhibited during environmental stress situations. This mutant, IM222, was used to test the effects of USA300 WT versus the SAUSA300_1759 gene knockout on the clumping and bordering phenotype of C. elegans. This assay resulted in a lack of clumping and a longer duration prior to bordering observed in the gene knockout strain when compared to the USA300 WT strain, however, nematodes exhibited both phenotypes more rapidly. This difference was confirmed through complementation. This could indicate SAUSA300_1759 plays an important role in USA300's pathogenesis. Further research needs to be performed to establish promoter activation through human cell line co-culture to solidify these findings. Defining the exact role of this protein in the infection cycle of CA-MRSA strain USA300 will allow a better understanding of this organism's pathogenicity as well as provide new opportunities for the development of novel treatments for these infections.

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